Neuronal injury-induced expression and release of apolipoprotein E in mixed neuron/glia co-cultures: nuclear factor κB inhibitors reduce basal and lesion-induced secretion of apolipoprotein E
Section snippets
Cell cultures
Mixed neuron/glia cultures were prepared from fetal 18-day-old Sprague–Dawley rat embryos (Harlan, Indianapolis, IN, USA). In brief, brain tissue was dissected on ice, and the cerebral cortices were freed of meninges and dissociated in 5 ml of 0.05% trypsin/0.53 mM EDTA for 15 min at 37°C. Trypsin was then neutralized by addition of DNase (200 U/ml) and minimum essential medium (MEM) containing 10% fetal bovine serum, and the cell suspension was centrifuged at 200g for 4 min. The pellet was
Characterization of the model: N-methyl-d-aspartate-induced neuronal death and glial proliferation
We first confirmed that exposure of mixed neuron/glia cultures to NMDA induced a concentration-dependent neuronal death, measured by LDH release. The neurotoxic effect of NMDA was maximal at 100 μM (15–25% of maximal cell death; Fig. 1A). Next, we studied whether NMDA-induced neuronal death was accompanied by glial proliferation. As seen in Fig. 1B, the number of proliferating cells in control wells was highest at days 1 and 2 and then declined. NMDA treatment did not induce significant
Discussion
The APOE4 allele confers a high susceptibility for late-onset and sporadic AD and a poor outcome after head trauma promoting amyloid deposition, therefore enhancing the risk for AD.25., 43., 54., 55. However, APOE4 is neither necessary nor sufficient for developing AD, since this risk factor accounts for only 30–50% of the late-onset cases.18 Environmental or other biological factors may influence the onset of the disease. For instance, apoE expression, independent of genotype, could be a
Acknowledgements
J.S. is a recipient of a grant from the Spanish Ministry of Education. We gratefully thank Dr Neil DeLapp for help in western and northern quantitation, and Pamela Edmonds for excellent editorial work.
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V. Petegnief and J. Saura contributed equally to this work.